Microorganisms are involved in the manufacture of food and feed products including most dairy products. Bacterial cultures, in particular cultures of bacteria generally classified as lactic acid bacteria, are essential in the making of all fermented milk products, cheese and butter. However, cultures of certain non-bacterial microorganisms, e.g. certain yeasts and fungi, are used to process food and feed products. Cultures of these microorganisms are often referred to as starter cultures and impart specific features to various dairy products by performing a number of functions. Starter cultures are widely used in a variety of industries such as, the diary industry as well as in the wine manufacturing industry, and the juice manufacturing industry, the meat processing industry.
Cultures-of microorganisms also find important uses in the biopreservation of food-stuffs (Andersen et al., 1997).
Commercial dairy starter cultures are generally composed of lactose and citric acid fermenting bacteria. Lactic acid bacteria designate a group of Gram positive, non-motile, microaerophilic or anaerobic bacteria that ferment sugar with the production of acids including lactic acid. Industrially some of the most useful lactic acid bacteria include Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species.
Commonly used dairy starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30° C. and thermophilic organisms having optimum growth temperatures in the range of about 35° C. to about 45° C. Examples of organisms belonging to the mesophilic group include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar diacetylactis and Lactobacilius paracasei subsp. paracasei. Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus. 
Dairy starter cultures are also classified according to their specific species composition and preferred industrial use. A pure starter culture comprises only a single specie whereas a mixed culture comprises two or more different species. Starter cultures are often categorized according to the temperature at which they display optimal growth or maximal enzymatic activity. Mesophilic starter cultures typically have an optimum temperature of about 30° C., whereas thermophilic cultures have an optimum temperature of about 35-45° C. (Nielsen and Ullum, 1999). Examples of commercial mesophilic mixed cultures include:                “O-culture” comprising Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.         “D-culture” comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar diacetylactis.         “L-culture” comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc species.        “LD-culture” comprising Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc species.        
An O-culture is used to make cheese without holes (Cheddar, Cheshire, Feta). A D-culture is used to make butter. A L-culture is used to make cheese with smallholes (e.g., cottage cheese) and curdled milk products with low CO2-production. A LD-culture is used to make cheese with normal hole sizes, curdled milk products (junket) and sour butter. Commercially, LD-cultures are currently one of the most used mixed cultures.
Examples of commercial thermophilic mixed cultures include:                “Yoghurt culture” comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and        “Thermophilic cheese culture” comprising Streptococcus thermophilus and Lactobacillus helveticus.         
A Yoghurt culture is used to make yoghurt and special Italian cheeses, Thermophilic cheese culture is used to make Emmentaler cheese and special Italian cheeses.
In addition, species of Propionibacterium are frequently used as dairy starter cultures, particularly in the manufacture of cheese. Also organisms belonging to the Brevibacterium genus and the Bifidobacterium genus are commonly used as food starter cultures.
Another group of microbial starter cultures is fungal cultures, including yeast cultures and cultures of filamentous fungi, which are useful in the manufacture of certain types of cheese and beverage. Examples include Penicillium roqueforti, Penicillium candidum, Geotrichumcandidum, Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae. 
Starter cultures are also widely used in the meat processing industry, e.g. for the manufacturing of various .sausages and salamis.
Commercial starter cultures are commonly be distributed as frozen cultures. At the low temperature the frozen cultures, most metabolic activities in the cell cease and cells can be maintained in this suspended, but viable, state for extended periods.
Concentrated frozen cultures are commercially very interesting since the cultures can be inoculated directly. into the, production container. By using concentrated frozen cultures the end-user avoids the otherwise obligatory, time-consuming intermediary fermentation step during which the starter culture is amplified, and the end-user reduces the risk of contamination significantly. Concentrated cultures, may be referred to as DVS—direct vat set™ cultures.
As an alternative to concentrated frozen cultures, concentrated freeze-dried DVS™ cultures may be prepared. These cultures have an additional advantage in that they can be shipped without refrigeration.
In general, possible damaging effects of freezing and thawing on the viability of living cells has been ascribed to cell dehydration and the formation of ice crystals in the cytosol during freezing.
A number of cryoprotective agents have been found to effect the concentration of the cytosol in a controlled and minimally injurious manner so that ice crystallization in the cytosol is precluded or minimized during freezing.
An article by F. J. Chavarri et al. (Biotechnology letters, vol 10, 1, 11-16 (1988), “Cryoprotective agents for frozen concentrated starters from non-bitter Streptococcus Lactis strains”) describes the storage viability of a frozen pure Streptococcus lactis culture may be improved by addition of 5% lactose or 5% sucrose. The lactose or sucrose worked as cryoprotective agents. Streptococcus lactis is a former name of Lactococcus lactis subsp. lactis. 
Similarly, an article by R. Cárcoba et al (Eur Food Res Technol (2000) 211,433 -437, “Influence of cryoprotectants on the viability and acidifying activity of frozen and freeze-dried cells of the novel starter strain Lactococcus lactis subsp. lactis CECT 5180”) describes that storage viability of a frozen pure Lactococcus lactis subsp. lactis culture could be improved by addition of different cryoprotective agents such as sugars (lactose, sucrose and trehalose), glutamic acid and gelatine.
The viability of freeze-dried cultures may also be improved by use of cryoprotective agents. For instance EP259739 describes a number of different cryoprotective agents for freeze-dried cultures.
There have been various approaches to provide cryoprotection such as carbonhydrates, proteins and certain surface active agents.
In general relatively large amounts of cryoprotective agents are required in order to obtain the cryoprotective effect. While this presents an insignificant problem in some settings, it presents a significant problem for food processing industries where even a small undesired deviation in the taste of the fermented or processed product that is caused by the cryoprotective agent can be detrimental. We are not aware of any commercial available concentrated frozen cultures that contain significant amounts of cryoprotective agents.
Agents other than carbonhydrates, proteins and surface-active agents have been used to improve the stability of cultures at low temperature.
WO 00/39281 describes the use of IMP and compounds involved in the biosynthesis of DNA synthesis to stabilize the metabolic activity of a liquid starter cultures, rather than the stability of frozen or freeze-dried cultures.
WO 00/19817 describes a cryoprotective composition, in which a combination compounds rather than any single component is used for cryoprotection. The combination includes a calcium channel blocker, a cell nutrient matrix, water and adenosine. However, the use of pharmaceutical active compounds such as calcium channel blockers are not acceptable for food industry applications.
JP 05 308956 describes a culture medium for the culture of nitrite bacteria that comprises a high molecular polysaccharide in which a unit consisting of 1 molecule of alpha-Lrhamnose, 1 molecule of D-glucuronic acid and 2 molecules of D-glucose polymerized linearly and e.g. ATP. Nitritate bacteria cultured in this medium can be frozen and stored. There is no indication that components of the medium may function as a cryoprotective agent when added to concentrated cultures prior to freezing.
The need remains for effective cryoprotective agents that can be added to concentrated cultures used in the food industry.